Biosynthesis of Polyunsaturated Fatty Acids
Fatty acids are major components of lipids such as phospholipids and triacylglycerols. Fatty acids containing two or more unsaturated bonds are collectively referred to as polyunsaturated fatty acids (PUFAs), and are known to include arachidonic acid, dihomo-γ-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, etc. Various physiological activities have been reported for these fatty acids (non-patent document 1).
These polyunsaturated fatty acids are expected to find applications in various fields, but some of them cannot be synthesized in vivo in animals. This has led to development of methods for obtaining polyunsaturated fatty acids by culturing various microorganisms. Attempts to produce polyunsaturated fatty acids in plants have also been made. In such cases, polyunsaturated fatty acids are known to be accumulated as components of reserve lipids such as triacylglycerols, for example, in microbial cells or plant seeds.
Among the polyunsaturated fatty acids, arachidonic acid has attracted attention as an intermediate metabolite in the synthesis of prostaglandins, leukotrienes and the like, and many attempts have been made to apply it as a material for functional foods and medicaments. Furthermore, arachidonic acid is contained in breast milk so that it is important for the growth of infants, especially for the growth of fetal length and brain, and therefore, it also attracts attention in a nutritional aspect as a necessary component for the growth of infants as well as DHA (docosahexaenoic acid).
Arachidonic acid is biosynthesized by the pathway shown in FIG. 1. Specifically, arachidonic acid is produced through several chain elongation and desaturation steps from palmitic acid generated by de novo fatty acid synthesis. In this pathway, an elongase and Δ9 desaturase act on acyl-CoA. On the other hand, Δ12 desaturase, Δ6 desaturase and Δ5 desaturase are known to act on the acyl groups of phospholipids such as phosphatidylcholine (non-patent document 2). Thus, acyl transfer between acyl-CoA and phospholipids is required in the biosynthesis of PUFAs such as arachidonic acid. Without being limited to the biosynthesis of PUFAs, replacement of only fatty acids after biosynthesis of phospholipids is known as “remodeling” of phospholipids, and lysophospholipid acyltransferases (hereinafter referred to as “LPLATs”) are known to be involved in this reaction (non-patent document 3).
Biosynthesis of Triacylglycerols
Among reserve lipids, triacylglycerols are synthesized in vivo as follows. Glycerol-3-phosphate is acylated with glycerol-3-phosphate acyltransferase (hereinafter sometimes referred to as “GPAT”) at the hydroxyl group in the 1-position (α-position) to form lysophosphatidic acid (hereinafter sometimes referred to as “LPA”). LPA is a lysophospholipid containing only one acyl group, and is acylated with lysophosphatidic acid acyltransferase (hereinafter sometimes referred to as “LPAAT”) to form phosphatidic acid (hereinafter sometimes referred to as “PA”). This PA is dephosphorylated by phosphatidic acid phosphatase to form diacylglycerol, which is in turn acylated with diacylglycerol acyltransferase (hereinafter sometimes referred to as “DGAT”) to form triacylglycerol. Acyl-CoA: cholesterol acyltransferase (hereinafter sometimes referred to as “ACAT”) and lysophosphatidylcholine acyltransferase (hereinafter sometimes referred to as “LPCAT”) and the like are known to be indirectly involved in the biosynthesis of triacylglycerols.
Biosynthesis of Phospholipids
PA produced from LPA by the action of LPAAT as described above serves as a precursor in the biosynthesis of various phospholipids. For example, important phospholipids such as phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylglycerol (PG) are biosynthesized from PA. Thus, PA is not only an intermediate in lipid synthesis, but also an intracellular and intercellular lipid mediator having a very wide range of biological and pharmacological effects such as cell proliferation, platelet aggregation, smooth muscle contraction, promotion of cancer invasion, etc.
Lysophospholipid Acyltransferases
As described above, LPLATs are believed to be involved in PUFA biosynthesis. The LPLATs collectively refer to enzymes having the activity of introducing an acyl group into lysophospholipids, and include those having various names based on the specificity for the substrate, i.e., the molecular species of the lysophospholipid used as a substrate. One example is LPAAT that is involved in the synthesis of triacylglycerols and phospholipids using LPA as a substrate. Other lysophospholipids on which LPLATs act include lysophosphatidylcholine (LPC), lysophosphatidylserine (LPS), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), etc. Thus, the enzymes are called LPAAT, LPCAT, lysophosphatidylserine acyltransferase (LPSAT), lysophosphatidylinositol acyltransferase (LPIAT) and the like based on the molecular species on which they act. Each enzyme may specifically act on one lysophospholipid or multiple specific lysophospholipids. For example, LPLATs called as LPAAT include those acting on not only LPA but also LPC, LPE, etc.
Sequence Profile-Based Classification of Lysophospholipid Acyltransferases
LPLATs are classified as glycerophospholipid acyltransferases. The glycerophospholipid acyltransferases are thought to fall into three groups from amino acid sequence comparison, i.e., LPAAT family, MBOAT (membrane-bound O-acyltransferase) family and DGAT2 family (non-patent document 5). Enzymes belonging to the LPAAT family are commonly characterized by a membrane-bound domain and a sequentially conserved motif (LPAAT motif). The enzymes belonging to the LPAAT family members include LPAAT, GPAT, etc. Enzymes included in the MBOAT family are commonly characterized by a membrane-bound domain. The MBOAT family is known to include DGAT, ACAT and the like in addition to LPLAT. In animals or the like, some enzymes belonging to the MBOAT family are thought to be responsible for the remodeling reaction critical for membrane phospholipid synthesis.
LPLATs have been reported in a broad spectrum of organisms from unicellular organisms such as bacteria and yeast to higher organisms such as mammals. In yeast (Saccharomyces cerevisiae) belonging to fungi, SLC1 (YDL052C) and SLC4 (YOR175C) (herein sometimes referred to as “ALE1” or “LPT1”) are known as membrane-bound LPLAT genes (non-patent document 5). In animals, multiple LPLAT homologs are known to exist, including those responsible for the reaction of acting on LPA in the de novo triglyceride synthesis system to yield PA and those responsible for phospholipid remodeling (non-patent document 6).
In the lipid-producing fungus Mortierella alpina (hereinafter sometimes referred to as “M. alpina”), four LPLATs have been obtained, all of which belong to the LPAAT family (patent documents 1-3). However, no report shows that any LPLAT belonging to the MBOAT family has been obtained from M. alpina. 